Combination of an HMG-CoA reductase inhibitor and a farnesyl-pyrophosphate synthase inhibitor for the treatment of diseases related to the persistence and/or accumulation of prenylated proteins

ABSTRACT

The invention relates to the use of a hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and of a farnesyl-pyrophosphate synthase inhibitor, or of one of their associated physiologically acceptable salts, in the preparation of a composition, particularly a pharmaceutical composition, for use in the treatment of human or animal, pathological or nonpathological situations related to the accumulation and/or the persistence of prenylated proteins in cells, such as during progeria (Hutchinson-Gilford syndrome), restrictive dermopathy or physiological ageing.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 12/307,220, filed Dec. 31, 2008, which is aNational Stage entry of International Application No. PCT/FR2007/001144,filed Jul. 5, 2007, which claims priority to France Patent ApplicationNo. 06/06097, filed Jul. 5, 2006, the disclosures of the priorapplications are hereby incorporated in its entirety by reference.

The present invention lies in the field of the treatment of conditions,pathological or not, related to the accumulation and/or persistence incells of prenylated proteins.

The nucleus of eukaryotic cells is delimited by a double membrane withpores in it, the nuclear envelope, which controls the molecularexchanges between the two nuclear and cytoplasmic compartments. Thisenvelope partially isolates the content of the nucleus, that is to saythe genetic material and all the enzymatic machinery necessary for thefunctions of the nuclear genome.

The nucleus envelope consists of two concentric membranes, the externalmembrane, in continuity with the endoplasmic reticulum, and the internalmembrane. The latter is bordered on its internal face by a densefibrillar mesh called the nuclear lamin. This is a protein latticecomposed essentially of lamin polymers and associated proteins. Invertebrates, there are two sub-classes of lamins: type A lamins (laminsA and C), and type B lamins (lamins B1, B2 and B3), which allparticipate in the production of the lamin. The latter is held in placeby association with other proteins, fixed to the internal membrane ofthe nuclear envelope (for journal, Gruenbaum et al 2005).

Lamins are proteins in the form of filaments belonging to the family ofintermediate filaments (type V), which all have a common structure: ashort N-terminal globular segment (the head) separated from anotherC-terminal globular segment (the tail) by a long central domainorganised in several alpha helixes (the rod domain). The globular tailcontains in particular a nuclear location signal (NLS) allowingaddressing of the nucleus after synthesis. The central domain allows theassociation of two parallel lamin molecules and their organisation infilaments by the association of dimers in opposite orientations. Thisstructure confers very resistant mechanical properties on them.

Only the A-type lamin and the B-type lamins undergo maturation after thesynthesis of a precursor (for journal, Gruenbaum et al 2000). The C-typelamin is directly synthesised in its mature form.

The precursor of the A-type lamin and the B-type lamins terminates in acharacteristic CaaX unit (C is a cysteine, a an amino acid with anon-charged aliphatic chain and X any amino acid, here a methionine, forjournal, Levy & Cau 2003).

The C-terminal CaaX unit allows the fixing of a fatty acid (in general aC15 fatty acid, farnesyl) by virtue of a farnesyl-transferase. Thisprenylation (the farnesyl unit derives from a C5 base aliphatic unitcalled isoprene) enables the prelamins to be inserted in the membrane ofthe endoplasmic reticulum after their synthesis in cytosol. They undergotherein the action of an endoprotease, itself inserted in the envelopemembrane of the reticulum and whose active site is cytosolic. Thespecific endoprotease of the prelamin A is Face1 (or ZMPSTE24 (ZincMetallo-Protease homologue of yeast STE24), while Face2 (or Rce1,Ras-converting enzyme) is specific to the B prelamins. These enzymescatalyse the hydrolysis of the peptide bond between the cysteine and thefollowing amino acid (aliphatic), shortening the prelamins by 3 aminoacids. The carboxyl end of the farnesylated cysteine is then recognisedby an isoprenylcysteine-carboxymethyl transferase (ICMT), which fixes amethyl group thereto by esterification.

Only the maturation of the prelamin A continues with a secondendoproteolytic cleaving by Face1, which releases a farnesyl-peptide of15 amino acids and a mature lamin A. This lamin A, which no longerincludes the fatty acid, becomes soluble, and is imported into thenuclear by virtue of its nuclear location signal, and is located in thenuclear lamin itself as well in the rest of the nucleus compartment,constituting a veritable nuclear skeleton (Gruenbaum et al 2005). The onthe other hand mature B lamin still has at the C-terminal end itsfarnesylated and methylesterified cysteine. It therefore remainsinserted in the envelope membrane of the reticulum, and then in thenucleoplasmic face of the nuclear envelope, and has its locationexclusive to the nuclear lamin, and the internal membrane of the nuclearenvelope where it is anchored.

The term prenylation should be taken to mean the fixing to the thiolgroup of a cysteine either of a farnesyl chain of 15 carbon atoms, andfarnesylation is then spoken of, or a geranylgeranyl chain of 20 carbonatoms, and geranylgeranylation is then spoken of (Reid et al 2004), orof any other derivative of isoprene.

The farnesylation, catalysed by the farnesyl-transferase (FTase), whichrecognises the C-terminal consensus sequence (CaaX), preferentiallyfixes a farnesyl group to the cysteine residue of the unit.

Gernaylgeranylation is the fixing by geranylgernayl-transferase (GGTase)of a geranylgeranyl group to the cysteine residue of the unit.

These fatty acids result from the biosynthesis method which, usinghydroxymethyl-glutaryl-Coenzyme A, is used by the cells formanufacturing in particular cholesterol, steroids, the haem ofhaemoglobin and ubiquinones (Hampton et al 1996).

The family of prenylated proteins comprises approximately 300 members inthe human genome, the majority of which can be identified by theC-terminal unit CaaX (Reid et al 2004). The proteins of the familiesRas, Rho, Rab (Leung et al 2006), certain proteins fulfilling an importfunction to the mitochondria (HDJ2), some mitotic proteins (CENPE,CENPF) are in particular prenylated (Winter-Vann & Casey 2005). Ingeneral, if in the CaaX unit, X is a serine, a methionine, a cysteine,an alanine or a glutamate, the preferentially grafted isoprenoid isfarnesyl. If X is a leucine, the CaaL unit will preferably recognised bythe GGTase, which will catalyse the transfer of a geranylgeranyl group(Basso et al 2006). It is probable that other groups derived fromisoprene can also be fixed to this cysteine, although this is notdescribed in the literature.

In humans, there exist three lamin genes. The LNMA gene, situated at1q21.2-q21.3 (Wydner et al 1996), gives the lamins A and C byalternating splicing. The LMNA gene is composed of 12 exons. The startof exon 1 codes the N-terminal globular end common to lamins A and C;the end of exon 1 and up to the start of exon 7 code the central helicalpart; finally, the other exons code the C-terminal globular end (Levy &Cau 2003).

In fact, the gene codes for 4 differently spliced products, the C laminsand the prelamin A of which are the two main ones (Lin & Worman 1993).The differential production of lamins A and C is done by the use of analternative splicing site at exon 10 of the pre-messenger, so that thelamin C is coded by exons from 1 to 10 and the lamin A is coded by exonsfrom 1 to 9, the first 90 pairs of bases of exon 10, and exons 11 and 12(A-specific lamin).

Consequently the prelamin A and the lamin C peptides are identical atthe first 566 amino acids, the C-terminal ends of the C lamins and theprelamin A next containing respectively 6 and 98 specific amino acids.

The type-B lamins comprise three different proteins (Shelton et al1981): lamins B1, B2 (the two most represented isoforms) and B3. TheLMNB1 gene is situated at 5q23.3-q31.1 and comprises 11 exons coding thelamin B1 (Lin & Worman 1995). The LMNB2 gene is located at 19p13.3 andcodes for the lamins B2 and B3 by an alternating splicing mechanism(Biamonti et al 1992).

The type-B lamins are expressed constituently in all the cells as fromthe first development stages, while the type A lamins are in generalabsent in the embryonic strain cells (Rober et al 1989, Stewart et al1987) and are expressed in all the differentiated somatic cells. Theirexpression is subject to regulations according to the tissue and duringlife (Duque et al 2006). It seems that their expression is notnecessary, since mice in which the lamin A expression has beenspecifically blocked, but which all the same express the lamin C and theother lamins, do not have any apparent phenotype (Fong et al 2006).

The lamins interact with a very high number of protein partners havingvery diverse functions; they are consequently involved in a large numberof nuclear processes, including DNA replication and repair, control oftranscription and splicing, organisation of the chromatin structure (forjournal, see Shumaker et al 2003, Zastrow et al 2004, Hutchison et al2004, Gruenbaum et al 2005). Alterations to the structure of the Lamingive rise to numerous human hereditary pathologies. They are due tomutations of the genes coding the lamins, or other proteins of thelamin. These pathologies have been grouped together under the genericterm laminopathies (Broers et al 2006, Mattout et al 2006). Recently,mutations in the genes of the enzymes responsible for the maturation oflamins (Face1 in particular) have been identified, giving rise topathologies also belonging to the group of laminopathies (Navarro et al2004 and 2005).

At the present time, the only pathology in humans associated withmutations of the LMNB1 or 2 genes is a leucodystrophy caused by acomplete duplication of the LMNB1 gene (Padiath et al 2006). A doubtremains on the potential implication of variation sequences found inLMNB2 in patients suffering from Barraquer-Simon syndrome (Hegele et al2006). However, it has been demonstrated in vitro by RNAi(RNA-interference) experiments (Harborth et al 2001), and in the murinmodel (Vergnes et al 2004), that type B lamins are essential for celldevelopment and integrity. This is because a lamin B1 deficiency causesperinatal mortality in mice. Moreover, the nuclei of the embryonicfibroblasts of the same LMNB1 deficient mice show remarkable alterationsin the nucleus morphology, close to those observed in patients carryingmutations of the LMNA gene. In addition, it has been shown recently thatB-type lamins are necessary to the formation of the division spindleduring mitosis, which tends to prove that their role is dynamic andmultiple during the cell cycle, not only restricted to maintenance ofthe architecture of the nucleus (Tsai et al 2006). On this latter role,a recent article demonstrates the structural function of the B-typelamins: cells artificially deprived of B1-type lamins have a “floating”nucleus in the cell, which turns on itself (Li et al 2007). Thefunctional redundancy existing between the two lamins B1 and B2 is nodoubt also a direct reflection of their indispensability, exerting ahigh selection pressure and masking the effect of any mutations of thesequence of corresponding genes.

The functional alterations in the A/C lamins, due to mutations of theLMNA gene, give rise to at least 15 disorders including very diversepathologies in a clinical spectrum ranging from mild forms, affecting atissue in an isolated fashion, to systemic forms that are fatal in theperinatal period.

Many mutations of the LMNA gene appreciably modify the assembly ofproteins in the nuclear envelope and disturb their functioning. In thecells of various tissues, the morphology of the nuclei is altered: theyoften have hernias that extrude genetic material in the cytoplasm(Goldman et al 2004).

The proteins normally associated with the nuclear envelope, the Blamins, certain proteins of the nuclear pores and the LAP2 proteins areabsent from the periphery of these hernias.

These morphological anomalies are followed by functional alterations,which end up by causing cell deaths. Among all the pathologies groupedtogether under the term laminopathies, only those relating to theabnormal accumulation of a prenylated form of protein relate to thepresent invention.

These are mainly the Hutchinson-Gilford syndrome, or Progeria (DeSandre-Giovannoli et al 2003, Eriksson et al 2003), and restricteddermopathy (Navarro et al 2004). In these 2 syndromes, thephysiopathological cause is an accumulation and persistence in the cellsof the patients of non-matured farnesylated prelamin.

Restrictive dermopathy, fatal around the natal period, is characterisedby clinical signs that are almost all the consequence of a cutaneousdeficit that restricts movements in utero. This pathology is very rare.The skin is rigid and tensioned, and yields in places, causing forexample tears at the armpits or neck. The eyelashes, eyebrows and skinhair are absent or very sparse. Hydramnios is often present, and areduction in foetal movements is signalled as from the sixth month ofpregnancy. At the skeletal level, radiography reveals contractions atall of the joints, deformed feet, thin, dysplastic and bi-partitedclavicles, ribbon-shaped ribs, tubular long bones of the arms anddemineralisation at the cranium. Transmission of fatal restrictivedermopathy is autosomal recessive.

LMNA and ZMPSTE24/Face1 mutations have been reported for this pathology(Navarro et al 2004). In both cases, the physiopathological mechanism isthe same: the prelamin A cannot mature (zero Face1 mutation ordisappearance of the cleavage site by mutation of the prelamin A), andremains farnesylated, and therefore inserted in the nuclear membrane.The accumulation and persistence in the cells of these abnormalprecursors, which probably prevent normal interaction of the lamins Band C with their partners, causes death of the cells and, very soon, ofthe patient. It has been clearly demonstrated that it is indeed thepersistence of the farnesylated group rather than the absence of maturelamin A, as might have been thought at first, that is responsible forthe cell toxicity (Fong et al 2004).

In April 2003, from cross-checking the symptoms common to acromandibulardysplasia and some diseases resulting in premature ageing, the inventorsshowed that Progeria, the most typical and most serious form ofpremature ageing, results from a mutation of the LMNA gene (DeSandre-Giovannoli et al 2003). Children afflicted by this illness, alsoreferred to as Hutchinson-Gilford syndrome, suffer from acceleratedageing, up to ten times more rapid than that of a normal individual, andhave a life expectancy that does not exceed 13 years. In France, onechild out of approximately six million is affected. The symptoms arecutaneous ageing, baldness, reduction in the size of the jaw andproblems related to old age, for example stiffness in the joints andcardiovascular disorders. The latter, such as myocardial infarction oratherosclerosis, are often the cause of death.

The mutation involved, situated at exon 11 of the LMNA gene, activates acryptic splicing site of the pre-RNAm, leading to a deleted RNAm of 150nucleotides (De Sandre-Giovannoli et at 2003, Eriksson et al 2003). Thisdeleted RNAm is translated into an abnormal prelamin A, progerin, whichcannot be matured into normal lamin A: the absence of 50 amino acids ofexon 11 comprising the protease recognition site blocks the secondcleavage of the progerin, the C-terminal end of which keeps itsfarnesylated group. It therefore remains inserted in the nucleoplasmicface of the nuclear envelope, which has characteristic alterations,hernias of the nucleoplasm in the cytosol and abnormalities in thedistribution of the peripheral heterochromatin (Goldman et al 2004).Here also, it is the persistence of the farnesylated group, moreovernecessary for anchoring to the envelope membrane of the reticulum inwhich there are located some of the enzymes responsible for maturation(cleavages, methylation) which is responsible for the cell toxicity ofthe progerin (Fong et al 2004).

These systemic pathologies have the particularity of being associatedwith the premature appearance of signs normally related to ageing. Theircommon physiopathological characteristic is to generate a prenylatedlamin, with the consequences described.

Two recent studies have shown that a reduction in the intranuclearaccumulation of the farnesylated prelamin, truncated or not, effectivelyprevents the appearance of the cell phenotype. The first was carried outon the progeroid murin model deficient in Face1 protease (Pendas et al2002). When they are crossed with mice expressing half the amount oflamin A (LMNA+/− mouse), the effects of the absence of Face1 are less(Verela et al 2005). The second study shows that the treatment of cellsof HGPS patients with morpholino (antisens oligonucleotides) targetingthe cryptic splicing site does away with the mutant phenotype (Scaffidi& Mistelli 2005).

Several recent studies (Scaffidi & Mistelli 2006, Cao et al 2007) showthe involvement of lamin A in the physiological ageing process. Inparticular, it has been demonstrated that, during physiological ageing,an aberrant lamin A accumulates over time at the periphery of the cellnucleus. This aberrant lamin is fact progerin, the cell, during itsnormal life and functioning accidently using from time to time thecryptic splicing site of exon 11, the progerin produced accumulateslittle by little under the lamin. Finally, the “normal” aged cell maypresent hernias characteristic of a laminopathy caused by theseaccidental splicing events, which cause its death.

It appears that identical molecular mechanisms are firstly responsiblefor the signs of premature ageing in individuals suffering from Progeriaand secondly, at a much lower level, are involved in the physiologicalageing of individuals not carrying mutations.

There exist in the prior art two therapeutic approaches described forimproving the cell phenotype caused by the pathological production ofprogerin. The first of these solutions is quite simply to prevent theuse by the spliceosome of this cryptic splicing site in exon 11, by“masking” it by treatment with an antisens oligonucleotide (Scaffidi &Mistelli 2005), or with a retrovirus producing an siRNA (Huang et al2005). The results are promising in vitro, but it is a case here of“gene” therapy, and the development of a medication around this approachis necessarily long and complicated, with all the drawbacks related tothe vectorisation of the OASs in order to obtain an in vivo effect. Thesecond solution consists of inhibiting the farnesyl-transferase, theenzyme that catalyses the transfer of the farnesyl group on theprelamins from farnesyl-pyrophosphate. When such inhibitors (FTI) areused, a “normal” nuclear envelope is only partially restored on HGPScells (Progeria) in culture, and the survival of RD mice (KO ZMPSTE24)is improved (Glynn & Glover 2005, Capell et al 2005, Toth et at 2005,Fong et al 2006).

However, blocking and farnesylation may cause a compensatorygeranylgeranylation (Bishop et al 2003).

In addition, it has been reported recently that FTIs cause a stoppage ofthe cell cycle by blocking the proteasome (Demyanets et al 2006, Efuet &Keyomarsi 2006). Thus the treatment no doubt causes an accumulation inthe nucleoplasm of progerin probably ubiquitinylated not degraded by theproteasome.

In addition, recent works report that the reduction in the level offarnesylation of the progerin in vivo is very low, around 5% (Young etal 2006), which does not suffice to explain the restoration in thenuclear morphology observed in vitro.

Finally, the FTIs are specific to only one of the protein prenylationroutes and cannot be envisaged as global inhibitors of post-translationprenylations.

Moreover, it is reported that the total absence of one of the enzymes ofthis route, mevalonate-kinase, is fatal during infancy (homozygotemutation loss of function of the gene coding for this enzyme, a syndromereported by Hoffmann et al 2003).

After lengthy research, the inventors have shown that the association ofan inhibitor of hydroxymethylglutaryl-coenzyme A reductase (the statinfamily) and an inhibitor of farnesyl-pyrophosphate synthase(amino-biphosphonates family, NBP), or of one of their physiologicallyacceptable salts, is an effective treatment for conditions, pathologicalor otherwise, related to the accumulation and/or persistence in cells ofprenylated proteins, in that it blocks all the protein prenylationchannel, both C15 and C20, or in the non-characterised forms. Theinventors have also found that the association of an inhibitor ofhydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and an inhibitor offarnesyl-pyrophosphate synthase has a synergetic effect of therestoration of the normal phenotype in fibroblasts of patients affectedwith Progeria. The effect of the association is appreciably greater thanthe effect of one or other of the inhibitors used individually.

The use of the association on cells of patients suffering from Progerialeads to an inhibition of the prenylation of proteins, and therefore tothe appearance of non-farnesylated prelamin A and the improvement ofnuclear symptoms.

The results make it possible to envisage the use of an inhibitor ofhydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and an inhibitor offarnesyl-pyrophosphate synthase, or one of their physiologicallyacceptable salts, in the preparation of a composition, in particular ofa pharmaceutical composition, intended for the treatment of conditions,pathological or otherwise, related to the accumulation and/orpersistence of prenylated proteins in cells.

Thus an object of the invention is the use of at least one inhibitor ofhydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and at least oneinhibitor of farnesyl-pyrophosphate synthase, or one of theirphysiologically acceptable salts, in the preparation of a composition,in particularly a pharmaceutical composition, intended for the treatmentof conditions, pathological or otherwise, related to the accumulationand/or persistence of prenylated proteins in cells.

It is also within the scope of the invention to use compounds that areboth inhibitors of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductaseand inhibitors of farnesyl-pyrophosphate synthase.

Advantageously, the composition according to the invention can beintended for the treatment of conditions, pathological or otherwise,related to the accumulation and/or persistence in cells of farnesylatedand/or geranylgeranylated proteins.

Very particularly, the composition according to the invention can beintended for the treatment of conditions, pathological or otherwise,related to the accumulation and/or persistence in cells of progerin,even more particularly to the treatment of conditions related to theaccumulation and/or persistence in cells of farnesylated prelamin A,whether or not this be truncated or modified.

In particular, since it is accepted that physiological ageing involvesthe accumulation of progerin in cells during life and that progerin isconcentrated in particular in the mesenchymatous cells, the compositionaccording to the invention can be intended to prevent the effects ofcell ageing, in particular with regard to the skin and/or the vascularendothelium.

The composition according to the invention can be intended for thetreatment of any living being, human or animal, particularly for theprevention of the effects of cell ageing. The composition thereforefinds an application both in human medicine and in veterinary medicine.

According to the invention, any inhibitor of farnesyl-pyrophosphatesynthase or one of its physiologically acceptable salts can be used inthe preparation of the composition according to the invention.

The physiologically acceptable salts may for example be salts formedwith hydrochloric, bromhydric, nitric, sulphuric or phosphoric acids,carboxylic acids such as for example acetic, formic, propionic, benzoic,maleic, fumaric, succinic, tartric, citric, oxalic, glyoxylic oraspartic acids, alkane sulphonic such as methane or ethane sulphonicacids, or arylsulphonic such as benzene or para-toluene sulphonic acids.

Particularly inhibitive of farnesyl-pyrophosphate synthase may be one ofthe members of polyphosphonate family, particularly aminobisphosphonates(NBPs), or one of its physiological acceptable salts.

Polyphosphonates are synthetic molecules very much used in the treatmentof osteoporosis and bone regeneration.

The term phosphonate applies to molecules very similar to phosphate:

The core of the bisphosphonates (BPs) is equivalent to a P—O—P bond asin ATP for example, but where the oxygen is replaced by a carbon. Thisconfers special stability on these molecules.

A simple bisphosphonate with the equivalent ADP, the two phosphategroups (O₃P—) being replaced by the bisphosphonate group.

The central carbon, unlike oxygen in phosphates, may also be involved intwo bonds, and it is the nature of the groups grafted onto this carbonthat makes the specificity of bisphosphonates.

When the “lateral” chains (R1 and R2) comprise an amine function (NH),or more generally one or more nitrogen atoms, amino bisphosphonate, orNBP, is spoken of.

Naturally other substituents can be fixed to the oxygens.

Pyrophosphoric acid, or pyrophosphonate in solution (PPi)

is used in many metabolic reactions as a substrate transporter, and isrestored at the end of the reaction. One of the metabolic routes usingmolecules coupled to pyrophosphate is that of protein prenylation.

The grafting of isopentenyl-PP (C5 base unit) onto a geranyl-PP (C10) inorder to give farnesyl-PP, a reaction catalysed by the enzymefarnesyl-pyrophosphate synthase (FPS), releases a PPi.

It is this step that is specifically inhibited by the NBPs.

In this regard and by way of example, the aminobisphosphonate(farnesyl-pyrophosphate synthase inhibitor) can be chosen from

-   -   alendronic acid or its ionic form, alendronate;    -   clodronic acid or its ionic form, clodronate;    -   etidronic acid or its ionic form, etidronate;    -   ibandronic acid or its ionic form, ibandronate;    -   medronic acid or its ionic form, medronate;    -   neridronic acid or its ionic form, neridronate;    -   olpadronic acid or its ionic form, olpadronate;    -   pamidronic acid or its ionic form, pamidronate;    -   risedronic acid or its ionic form, risedronate;    -   tiludronic acid or its ionic form, tiludronate;    -   zoledronic acid or its ionic form, zoledronate;    -   4-N,N-dimethylaminomethane diphosphonic acid or its ionic form,        dimethylaminomethanediphosphonate;    -   α-amino-(4-hydroxybenzylidene)diphosphonate.

Preferentially according to the invention, it is preferred to usezoledronic acid (also called zolendronic acid) or its ionic form,zoledronate (also called zolendronate).

According to the invention, any inhibitor of HMG-CoA reductase, or oneof its physiological acceptable salts, can be used in the preparation ofthe composition.

In particular the inhibitor of HMG-CoA reductase can be a molecule inthe family of statins, whether it be liposoluble or hydrosoluble, or oneof its physiologically acceptable sales.

Statins have been revealed in mushrooms. They have an inhibitingactivity for HMG-CoA reductase, a key enzyme in the biosynthesis ofcholesterol and steroids, which catalyses the reduction of thehydroxymethylglutarate coupled to the coenzyme A in mevalonic acid(mevalonate in solution). This inhibition is provided by theirstructural analogy with the skeleton of hydroxymethylglutarate. Themetabolic route involved is certainly that of the biosynthesis ofcholesterol, but it is also that of the synthesis of the prenyl groups,polymers of the base unit with 5 isoprene carbons used for modifyingapproximately 300 proteins in the cells and attaching to them alypophilic tail, allowing in particular their anchoring in themembranes.

The main polyprenes, all issuing from pyruvate and HMG-CoA, are geranyl(C10), farnesyl (C15) and geranylgeranyl (C20).

All the statins are hepatoselective overall, but not all have the samemode of entry into the cells. This is because pravastatin androsuvastatin are both hydrophilic, and therefore hydrosoluble, unlikeall the others, which are lipophylic, and therefore can diffuse freelythrough the plasmic membranes (lipid bilayers), which no doubt explainstheir greater toxicity. Hydrosoluble statins need a specific transporterfor entering the cell, Organic Anion Transport 3, or OAT3, or SLC22A8(Takedaa et al 2004).

They are very much used for the treatment of hypercholesterolaemia, andtheir secondary effects, which are rare, are well characterised. Theseare in particular cases of rhabdomyolysis (1% to 7% of cases accordingto the molecule used, Evans et al 2002), the precursory sign of which,muscular pain in the patient being treated, gives rise to the immediatestoppage of the treatment.

In this regard and by way of example, a statin can be chosen fromatorvastatin, simvastatin, pravastatin, rivastatin, mevastatin (orcompactin), fluindostatin, velostatin, fluvastatin, dalvastatin,cerivastatin, pentostatin, rosuvastatin, lovastatin, pitavastatin, orone of their physiologically acceptable salts.

Lovastatin, pravastatin and simvastatin are molecules derived fromfungal metabolites, while the others (atorvastatin, cerivastatin,fluvastatin, pitavastatin and rosuvastatin) are entirely synthetic.Preferentially, according to the invention, privastatin is used, ahydrosoluble semi-natural statin.

Naturally it is possible according to the invention to use one or eventwo or more inhibitors of farnesyl-pyrophosphate synthase associatedwith one or even two or more inhibitors of HMG-CoA reductase.

According to a particular form of the invention, the composition can beintended for the treatment of conditions, pathological or otherwise,requiring inhibiting the prenylation of proteins. These pathologies maybe labelled or not, for example Costello or Noonan syndrome,cardio-fascio-cutaneous syndrome, or illnesses related to an abnormal orpersistent prenylation of Ras and signal transduction proteins.

According to yet another particular form of the invention, thecomposition can be intended for the treatment of conditions,pathological or not, presenting signs of ageing, whether it be natural,premature or accelerated. In particular in the case of signs ofdeterioration of the vascular endothelium (protection of the vascularendothelium), ageing of the skin, and bone lysis.

Preferentially, the composition according to the invention is apharmaceutical composition intended for the treatment of Progeria (HGPS,Hutchinson-Gilford Progeria Syndrome) and restrictive dermopathy (RD).

According to the invention, the inhibitor of farnesyl-pyrophosphatesynthase and the inhibitor of HMG-CoA reductase are advantageouslypresent in the composition at physiologically effective doses.

In general terms, the doses to be administered can be adapted by personsskilled in the art according to the patient, the pathology, theadministration mode, etc. Naturally, repeated administrations may beperformed, possibly in combination with other active ingredients or anyvehicle acceptable on a physiological level (buffers, saline solutions,isotonic solutions, with stabilisers, etc).

In general the daily dose of the inhibitors will be the minimum dose forobtaining the required therapeutic effect.

Compositions using an inhibitor of hydroxymethylglutaryl-coenzyme A(HMG-CoA) reductase and an inhibitor of farnesyl-pyrophosphate synthaseaccording to the invention can be formulated for the digestive orparenteral route.

The said compositions can also comprise at least one other activeingredient, in particular another therapeutically active ingredient, forsimultaneous or separate use or use staged over time.

According to the invention, the inhibitor hydroxymethylglutaryl-coenzymeA (HMG-CoA) reductase and the inhibitor of farnesyl-pyrophosphatesynthase can be used in the composition, in a mixture with one or moreexcipients or in inert vehicles, that is to say physiologically inactiveand non-toxic. Saline, physiological, isotonic, buffered, etc solutionscompatible with physiological usage and known to persons skilled in theart can for example be cited.

The compositions may contain one or more agents or vehicles chosen fromdispersants, solubilising agents, stabilisers, preservatives, etc.Agents or vehicles that can be used in formulations (liquid and/orinjectable and/or solid) are in particular methyl cellulose,hydroxymethyl cellulose, carboxymethyl cellulose, cyclodextrins,polysorbate 80, mannitol, gelatine, lactose, vegetable or animal oils,acacia, etc.

The compositions can be formulated in the form of injectable suspension,gels, oils, tablets, suppositories, powders, capsules, etc, possibly bymeans of galenic forms or devices providing prolonged and/or delayedrelease. For this type of formulation, an agent such as cellulose,carbonates or starches is advantageously used.

Administration can be achieved by any method known to persons skilled inthe art, preferably by oral route or injection, typically byintramuscular, intravenous or intra-peritoneal route,

Administration by oral route is preferred.

When it is a case of long-term treatment, the preferred administrationmethod will be sublingual, oral or transcutaneous.

FIG. 1 illustrates the results obtained in Western Blot on “normal”reference fibroblasts treated with increasing doses of a hydrosolublestatin (pravastatin P, 20 to 100 μm), and an aminobisphosphonate (NBPzoledronate Z, 20 to 100 μm) ((Tracks A to I, respectively P20/Z20,P20/Z60, P20/Z100, P60/Z20, P60/Z60, P60/Z100, P100/Z20, P100/Z60,P100/Z100)). Track J is a positive control for the presence of prelaminA (fibroblasts of patients DR), track K is the negative control, treatedwith the solvent alone (PBS).

FIG. 2 illustrates the results obtained at effective doses of each ofthe products.

FIG. 3 illustrates the superior effect obtained when the 2 products areadministered together.

FIG. 4 illustrates the action of the association of the 2 products onaged cells.

FIG. 5 illustrates the action of the association of the 2 products onmodel mice of Progeria (not synthesising the Face1 enzyme).

5 a: photograph representing mice aged 3 months and respectively wild,Zmpste24^(−/−) and Zmpste24^(−/−) treated.

5 b: weight of the wild mice (n=6), Zmpste24^(−/−) non-treated (n=7) andtreated (n=8), at 3 months.

5 c: survival curve (type Kaplan-Meier) showing the significantextension of the survival of treated females (n=7) (-⋄-) in comparisonwith non-treated mice (n=7) (-υ-).

5 d: tomography image, reconstituted in 3D, by μCT scanner of tibias ofmice treated and not treated (at the top). At the bottom, representationof the bone volume (on the left, a) and the number of trabeculae persquare millimetre (on the right, b).

5 e: quantification of nuclear abnormalities (%) on liver cells. On theleft, fluorescent marking of the nuclei with DAPI, on the right,counting of the number of abnormal nuclei.

The following examples serve to illustrate the invention without in anyway limiting it.

EXAMPLE 1 Synergetic Effect of the Association of an Inhibitor ofWater-Soluble Hydroxymethylglutaryl-Coenzyme A (HMG-CoA) Reductase andan Inhibitor of Farnesyl-Pyrophosphate Synthase on Cultures of NormalCells and Progeroids

A) Protocols

Cells and Cell Culture

The cell lines are either reference fibroblasts AG16409 from the CoriellInstitute, or fibroblasts from biopsies of patients suffering fromRestrictive Dermopathy. They are cultivated at 37° C. in 5% CO₂ in a P2room.

The normal complete culture medium is

-   -   RPMI (Invitrogen) supplemented with    -   Foetal calf syndrome 20% (Invitrogen)    -   L-Glutamine 200 mm (Invitrogen)    -   Penicillin/Streptomycin/Fungizone 1× mixture (Stock 100×,        Cambrex)

Harvesting of Cells

The cells are harvested by trypsinisation in the following manner(protocol for a large flask, 75 cm², BD Falcon):

The medium is aspirated;

The cells are washed with 10 ml of PBS 1× (Invitrogen), 10 ml;

5 ml of a solution of trypsin/EDTA 1× (Cambrex) is added

The flask is incubated for a period of approximately 6 minutes at 37°C., the time during which the cells detach;

The trypsin is inhibited by dilution in 15 ml of complete medium;

The cells are concentrated by centrifugation for 10 minutes at 1000revolutions per minute at 16° C.;

The concentrate is re-suspended in 2 ml of PBS 1×, and re-centrifugedunder the same conditions;

Either the cells are frozen for subsequent use or are transplanted fromthis washed concentrate.

Treatments

The pravastatin solution (water-soluble statin) used is prepared asfollows:

40 mg of pravastatin (Sigma Aldrich, P4498) is taken up in sterile waterin order to form a stock solution at 10 mM.

The final concentrations tested were 500 nM, 1, 5, 50 and 100 μM,obtained by diluting the stock solution in complete medium.

The solution of zoledronate (NBP) used is prepared as follows:

A stock solution of(1-hydroxy-2-imidazol-1-yl-phosphono-ethyl)phosphonic acid (0.8 mg/ml,Novartis) is adjusted to a concentration of 2 mM.

The final concentrations tested were 500 mM, 1, 5, 50 and 100 μM,obtained by diluting the stock solution in complete medium.

Western Blot

Preparation of Cells

For a Western blot experiment, the cells are treated as follows:

Approximately 7.5×10⁵ cells are seeded in a large flask and cultivatedunder the following conditions until there is almost confluence (4days).

At the end of 4 days, the cells are washed with PBS 1× and taken up inthe complete medium supplemented with the treatment.

The cells are incubated for the processing time (from 6 to 72 hours,sequentially or simultaneously) in the incubator at 37° C.

At the end of the processing, the cells are trypsinised (above protocol)and the concentrate obtained is stored at −80° C. until the proteins areextracted.

Extraction of the Proteins for Western Blot

The cell concentrate is taken up in 300 μl of lysis buffer

Triton X100  1% SDS 0.1% Sodium deoxycholate 0.5% NaCl 50 mM EDTA 1 mMTrisHCl pH 7.4 20 mM Protease inhibitor 1 pellet per 50 ml (Roche11697498001) Extemporaneously, there are added Sodium orthovanadate 1 mMPMSF 1 mM

The cells are exposed to sonication for twice 30 seconds (BrandsonSonifier Cell Disruptor B15)

The cell debris is centrifuged for 10 minutes as 10,000 revolutions perminute at 4° C.

The protein supernatant is stored at −80° C. until use.

The dosing of the proteins is carried out on defrosting.

Western Blot

Gel

An 8% acrylamide gel is conventionally used for detecting the differentforms of A/C-type lamins.

Acrylamide/bisacrylamide 37/1  8% TrisHCl pH 8.8 375 mM SDS 0.1% APS0.1% TEMED 0.01% 

A concentration gel is poured onto the separative gel

Acrylamide/bisacrylamide 37.5/1  3% TrisHCl pH 8.8 375 mM SDS 0.1% APS0.1% TEMED 0.01% 

The protein concentration of the samples is analysed, and aliquots areadjusted to 50 μg per tube in lysis buffer to qsp 15 μl.

5 μl of charge buffer is added to each sample.

SDS  4% TrisHCl pH 6.8 100 mM Glycerol 20% β-mercaptoethanol 20%Bromophenol blue traces

The samples are denatured by heating for 5 minutes at 95° C. anddeposited in the wells.

Migration takes place at 50 and then 100 volts, in a buffer

Tris-base 0.3% Glycine 1.44%  SDS 0.1%

Transfer

The transfer membrane (Hybon P, Amersham Biosciences) is prepared bysoaking in ethanol, followed by a bath for 5 minutes in sterile water,and 10 minutes in the transfer buffer:

Tris-base 12 mM Glycine 96 mM Ethanol 20%

The gel is moistened for 20 minutes in the transfer buffer and then thesandwich is mounted (Miniprotean system, Biorad).

The transfer takes place in general overnight, in a cold chamber, at 10volts.

The membrane is rinsed in PBS 1×, stored away from moisture, and used asit is for detection.

Detection

The membrane is incubated for 1 hour at room temperature in a saturationsolution:

Casein  10% Tween 20 0.1% PBS 1 X

It is rinsed for twice 10 minutes in a washing buffer (Tween 20 0.1%/PBS1×).

The primary antibody is diluted in the saturation buffer (details anddilution, see below immunomarking).

The membrane is incubated with the primary antibodies for 1 hour at roomtemperature under stirring.

At the end, it is rinsed 3 times with washing buffer and then washed for3 times 15 minutes with the same buffer.

The secondary antibody (system coupled to peroxydase, JacksonImmunoresearch) is diluted to 1/10000^(th) in saturation buffer.

The membrane is incubated with this solution for 30 to 45 minutes atroom temperature under stirring.

At the end, it is rinsed 3 times with washing buffer and then washed for3 times 15 minutes with the same buffer.

Detection is carried out with the ECL Plus Western Blotting System kitfrom Amersham Bioscience, according to the indications of the supplier.

After revelation, the membrane is exposed on Biomax MR film (Kodak), forthe time necessary to have a satisfactory signal.

Immunomarking

Preparation of Cells

A cell culture is trypsinised and the cells counted on a Neubauer cell.

Labtech-style culture wells (Nunc, ref 177399) are seeded, at the rateof 5×10⁴ cells per well.

The complete culture medium is supplemented by the treatment ortreatments (statin, NBP, or both), and the cells cultivated for an adhoc time.

At the end, the culture medium is aspirated and the wells dismantled.

The plates are washed in PBS 1×.

The cells are fixed in a paraformaldehyde 4% solution (in PBS) for 10minutes at room temperature.

Washing for 10 minutes in PBS 1× is carried out.

The cells are dehydrated by successive baths of 3 minutes in solutionswith an increasing ethanol percentage (70%, 90%, 100%, the latter bathbeing repeated).

After drying, the plates are stored at −80° C. until use.

Marking

After defrosting, the cells are incubated for 5 minutes at roomtemperature in a humid chamber in 50 μl of a permeabilisation solution:

PBS 1 X Triton X100 0.5% RNS  5% (Rabbit Normal Serum, Vector S5000)Protease inhibitor 1 pellet per 50 ml (Roche 11697498001)

3 pre-incubation baths each of 15 minutes are effected in 50 μl of theincubation solution:

PBS 1 X RNS 5% Protease inhibitor 1 pellet per 50 ml (Roche 11697498001)

The primary antibody is diluted to 1/100^(th) in 50 μl of incubationsolution and put in contact with the cells for 1 hour at roomtemperature in a humid chamber.

The primary antibodies used are of 2 types:

-   -   Anti-lamin mouse A/C(N-terminal side), clone 4A7, donated by G        Morris (Oswestry, UK)    -   Goat anti-prelamin A (15 aa C-terminal end), product SC6214,        Santa Cruz Biotechnology Inc.

Three rinsings in 50 μl of PBS 1× are carried out each for 15 minutes.

Incubation with the secondary antibody takes place for 1 hour in 50 mlof incubation solution at room temperature in a humid chamber. Thesecondary antibodies are of two types:

-   -   Anti-mouse donkey, Jackson Immunoresearch, dilution to        1/100^(th)    -   Anti-goat donkey, Jackson Immunoresearch, dilution to 1/200^(th)

Three rinsings in 50 μl of PBS 1× are carried out each for 10 minutes.

An incubation with 100 μl of DAPI 50 ng/ml solution (SERVA, ref 18860)is carried for 15 minutes at room temperature in a humid chamber.

Three rinsings in PBS 1× are carried out plate-holder tanks each for 5minutes.

A final rinsing is carried out for 5 minutes in a 0.1% solution ofTween20 in PBS.

Mounting

The cells are immersed in a drop of VectaShield (Vector), covered withan object-covering blade and observed on a fluorescence microscope(Leica DMR, Leica Microsystems), equipped with a coolSNAP (Princeton)camera system.

B) Results

B.1) Western Blot (FIG. 1)

“Normal” reference fibroblasts were treated with a water-soluble statin(pravastatin P, 20 to 100 μM), and with an aminobisphosphonate (NBPzoledronate Z, 20 to 100 μm) in association (tracks A to I, respectivelyP20/Z20, P20/Z60, P20/Z100, P60/Z20, P60/Z60, P60/Z100, P100/Z20,P100/260, P100/Z100). The Western blot shows the “appearance” of a bandcorresponding to the size of the immature (non-truncated) prelamin Aaccording to the increase in concentration of the two molecules, whichconfirms that farnesylation is necessary for the maturation of the laminA. This result shows that the blocking of the synthesis of thefarnesyl-PP at 2 points in the metabolic route is more effective than ablocking at a single point on the inhibition of the farnesylation of theprelamin A, at least ex vivo.

B.2) Dose And Duration Response in Immunohistochemistry (FIG. 2)

Dose-response and duration-response curves made it possible to determinea maximum efficacy by measuring 2 parameters on healthy reference cellson the one hand and then on cells of HGPS patients. The most effectivecombination of pravastatin (water-soluble)/zoledronate (NBP) is obtainedfor 1 μM of pravastatin for 24 hours and then zoledronate for 12 hours:on healthy cells, no toxicity is observed, whereas on HGPS cells (cellswith nuclear abnormalities), the number of “deformed” cells drops from75% to 40%. At the same time, the level of prelamin A obtained onhealthy cells is maximum.

B.3) Effect of the Immunohistochemistry Treatment (FIG. 3)

The combined action of the pravastatin and zoledronate shows betterefficacy since the level of prelamin A produced in treated healthy cells(estimated at 35%) is much higher in combination than if the moleculesare added alone (respectively 25% and 15%). On the other hand, the levelof deformed nuclei (the sign of toxicity on the healthy cells) isminimal (below 10%), less than it is on cells treated with pravastatinalone for example (approximately 12%).

Treatment: Pravastatin 100 μM for 12 hours, zoledronate 20 μM for 6hours.

B.4) Action on Aged Cells in Immunohistochemistry (FIG. 4)

According to the number of “passes” (the number of cell re-injections),and therefore the age of the cells, the proportion of abnormal nucleiincreases, which is a characteristic of the untreated HGPS cells. Ifthey are treated, this proportion is maintained, and even decreases alittle (less than 40% as against more than 80% in the cells treated witha placebo). Treatment: Pravastatin 1 μM for 24 hours, Zoledronate 1 μMfor 12 hours.

B.5) Conclusion

The pravastatin/zoledronate combination is effective at doses for whichalmost no effect is observed with the molecules administered separately.

The physiological effect of the blocking of the prenylation channel istherefore obtained with doses much lower than those used in singletreatment in articles published on cell cultures (Kusuyama et al 2006,10 μM of pravastatin alone on progenitors of vascular cells; Flint et al1997, 25 μM of pravastatin alone on neonatal rat muscle cells).

EXAMPLE 2 Effect of the Combination of an Inhibitor of Water-SolubleHydroxymethylglutaryl-Coenzyme A (HMG-CoA) Reductase and an Inhibitor ofFarnesyl-Pyrophosphate Synthase on a Mouse Model Having a ProgeroidSyndrome (FIG. 5)

The KO Zmpste24^(−/−) mice used here are those described in the articlecited by Varela et al 2005. They are raised in a breeding farm in acontrolled atmosphere, in a 12 hour day/12 hour night cycle, at atemperature of 20±2° C. and humidity of 50% and with access when desiredto food and water. The 2 molecules (Zometa 100 μg/kg/day and Pravastatin100 μg/kg/day) are dissolved in PBS 1× and injected by intraperitonealroute, daily, on mice aged from 1 month until their death. The controlsare wild mice of the same type, treated with PBS 1× alone. The weight ismeasured daily, and the survival of the mice is set out on a curve (FIG.5 b).

On the death of the mice, the tibias are dissected and analysed intomography by μCT scanner. The number of trabeculae per millimetre ismeasured by the method described in Hildebrand et al 1997 (FIG. 5 d).

The liver cells are sampled, fixed in paraformaldehyde at 4° C.,included in paraffin and analysed under confocal microscopy aftermarking of the chromatin with DAPI (4′6-diamidino-2-phenylindole). Thepercentage of abnormal nuclei is estimated by counting (FIG. 5 e).

The survival of the treated mice is greatly improved, and is maximum inparticular for females, with an extension of the average length of lifeby approximately 80% (FIG. 5 c). The clinical symptoms of illness areall considerably reduced compared with the individuals treated with PBSalone.

REFERENCES

-   -   Basso A D, Kirschmeier P, Bishop W R. Farnesyl transferase        inhibitors. J Lipid Res 47:15-31, 2006.    -   Biamonti G, Giacca M, Perini G, Contreas G, Zentilin L,        Weighardt F, Guerra M, Della Valle G, Saccone S, Riva S et al.        The gene for a novel human lamin maps at a highly transcribed        locus of chromosome 19 which replicates at the onset of S-phase.        Mol Cell Biol 12:3499-3506, 1992.    -   Bishop W R, Kirschmeier P, Baun C. Farnesyl transferase        inhibitors: mechanism of action, translational studies and        clinical evaluation. Cancer Biol Ther 2:S96-104, 2003.    -   Broers J L, Hutchinson C J, Ramaekers F C. Laminopathies. J        Pathol 204:478488, 2004.    -   Broers J L V, Ramaekers F C S, Bonne G, Ben Yaou R, Hutchinson        C J. Nuclear lamins: laminopathies and their role in premature        aging. Physiol Rev 86:9671008, 2006.    -   Cadinanos J, Varela I, Lopez-Otin C, Freije J M. From immature        lamin to premature aging: molecular pathways and therapeutic        opportunities. Cell Cycle 4:1732-1735, 2005.    -   Capell B C, Erdos M R, Madigan J P, Fiordalisi J J, Varga R,        Conneely K N, Gordon L B, Der C J, Cox A D, Collins F S.        Inhibiting farnesylation of progerin prevents the characteristic        nuclear blebbing of Hutchinson-Gilford progeria syndrome. Proc        Natl Acad Sci USA 102:12879-12884, 2005.    -   De Sandre-Giovannoli A, Bernard R, Cau P, Navarro C, Amiel J,        Boccaccio I, Lyonnet S, Stewart C L, Munnich A, Le Merrer M,        Levy N. Lamin A truncation in Hutchinson-Gilford progeria.        Science 300:2055, 2003.    -   Demyanets S, Kaun C, Pfaffenberger S, Philipp J. Hohensinner P        J, Rega G, Pammer J, Maurer G, Huber K, Wojta J.        Hydroxymethylglutatyl-coenzyme A reductase inhibitors induce        apoptosis in human cardiac myocytes in vitro. Biochem Pharmacol        71:1324-1330, 2006.    -   Duque G, Rivas D. Age-related changes in Lamin A/C expression in        the osteoarticular system: laminopathies as a potential new        aging mechanism. Mech Aging Dev 127:378-383, 2006.    -   Efuet E T, Keyomarsi K. Farnesyl and geranylgeranyl transferase        inhibitors induce GI arrest by targeting the proteasome. Cancer        Res 66:1040-1051, 2006.    -   Eriksson M, Brown W T, Gordon L B, Glynn M W, Singer J, Scott L,        Erdos M R, Robbins C M, Moses T Y, Berglund P, Dutra A, Pak E,        Durkin S, Csoka A B, Boehnke M, Glover T W, Collins F S.        Recurrent de novo point mutation in lamin A cause        Hutchinson-Gilford progeria syndrome. Nature 423:293-298, 2003.    -   Evans M, Rees A. The myotoxicity of statins. Cur Op Lipid,        13:415-420, 2002.    -   Flint O P, Masters B A, Gregg R E, Durham S K. HMG CoA reductase        inhibitor-induced myotoxicity: pravastatin and lovastatin        inhibit the geranylgeranylation of low-molecular-weight proteins        in neonatal rat muscle cell culture. Tox Appl Pharmacol        145:99-110, 1997.    -   Fong L G, Frost D, Meta M, Qiao X, Yang S H, Coffinier C, Young        S G. A protein farnesyltransferase inhibitor ameliorates disease        in a mouse model of progeria. Science, 311: 1621-1623, 2006.    -   Fong L G, Ng J K, Lammerding J, Vickers T A, Meta M, Cote N,        Gavino B, Qiao X, Chang S Y, Young S R, Yang S H, Stewart C L,        Lee R T, Bennett C F, Bergo M O, Young S G. Prelamin A and Lamin        A appear to be dispensable in the nuclear lamina. J Clin Invest        116:743-752, 2006.    -   Fong L G, Ng J K, Meta M, Cote N, Yang S H, Stewart C L,        Sullivan T, Burghardt A, Majumdar S, Reue K, Bergo M O, Young        S G. Heterozygosity for Lmna deficiency eliminates the        progeria-like phenotypes in Zmpste24deficient mice. Proc Natl        Acad Sci USA 101:18111-18116, 2004.    -   Glynn M W, Glover T W. Incomplete processing of mutant lamin A        in Hutchison-Gilford progeria leads to nuclear abnormalities,        which are reversed by farnesyltransferase inhibition. Hum Mol        Genet 14:2959-2969, 2005.    -   Goldman R D, Shumaker D K, Erdos M R, Eriksson M, Goldman A E,        Gordon L B, Gruenbaum Y, Khuon S, Mendez M, Varga R, Collins        F S. Accumulation of mutant lamin A causes progressive changes        in nuclear architecture in Hutchinson-Gilford progeria syndrome.        Proc Natl Acad Sci USA 101:89638968, 2004.    -   Gruenbaum Y, Margalit A, Goldman R D, Shumaker D K, Wilson K L.        The nuclear lamina comes of age. Nat Mol Cell Biol 6:21-31,        2005.    -   Gruenbaum Y, Wilson K L, Harel A, Goldberg M, Cohen M. Review:        nuclear lamins—structural proteins with fundamental functions. J        Struct Biol 129:313323, 2000.    -   Hampton R, Dimster-Denk D, Rine J. The biology of HMG-CoA        reductase: the pros of contra-regulation. Trends Biochem Sci        21:140-145, 1996.    -   Harborth J, Elbashir S M, Bechert K, Tuschl T, Weber K.        Identification of essential genes in cultured mammalian cells        using small interfering RNAs. J Cell Sci 114:4557-4565, 2001.    -   Hegele R A, Cao H, Liu D M, Costain G A, Charlton-Menys V,        Rodger N R, Durrington P N. Sequencing of reannotated LMNB2 gene        reveals novel mutations in patients with acquired partial        lipodystrophy. Am J Hum Genet 79:383-389, 2006.    -   Hildebrand T, Ruegsegger P. A new method for the model        independent assessment of thickness in three dimensional images.        J Microsc 185:67-75, 1997.    -   Hoffmann G F, Charpentier C, Mayatepek E, Mancini J,        Leichsenring M, Gibson K M, Divry P, Hrebicek M, Lehnert W,        Sartor K. Clinical and biochemical phenotype in 11 patients with        mevalonic aciduria. Pediatrics 91:915-921, 1993.    -   Huang S, Chen L, Libina N, Janes J, Martin G M, Campisi J,        Oshima J. Correction of cellular phenotypes of        Hutchinson-Gilford Progeria cells by RNA interference. Hum        Genet. 2005 Oct. 6; 1-7    -   Hutchinson C J, Worman H J. A-type lamins: guardians of the        soma? Nat Cell Biol 6:1062-1067, 2004.    -   Ji J Y, Lee R T, Vergnes L, Fong L G, Stewart C L, Reue K, Young        S G, Zhang Q, Shanahan C M, Lammerding J. Cell nuclei spin in        the absence of Lamin B1. J Biol Chem online, Aug. 5, 2007.    -   Kusuyama T, Omura T, Nishiya D, Enomoto S, Matsumoto R, Murata        T, Takeuchi K, Yoshikawa J, Yoshiyama M. The effects of HMG-CoA        reductase inhibitor on vascular progenitor cells. J Pharmacol        Sci 1001:344-349, 2006.    -   Leung K F, Baron R, Seabra M C. Geranylgeranylation of Rab        GTPases. J Lipid Res 47:467-475, 2006.    -   Lévy N, Cau P. Anomalies du noyau et maladies. Pour la Science        313:2-7, 2003.    -   Lin F, Worman H J. Structural organization of the human gene        (LMNB1) encoding nuclear lamin B1. Genomics 27:230-236, 1995.    -   Lin F, Woman H J. Structural organization of the human gene        encoding nuclear lamin A and nuclear lamin C. J Biol Chem        268:16321-16326, 1993.    -   Maraldi N M, Squarzoni S, Sabatelli P, Capanni C, Mattioli E,        Ognibene A, Lattanzi G. Laminopathies: involvement of structural        nuclear proteins in the pathogenesis of an increasing number of        human diseases. J Cell Physiol 203:319-327, 2005.    -   Mattout A, Dechat T, Adam S A, Goldman R D, Gruenbaum Y. Nuclear        lamins, diseases and aging. Cur Op Cell Biol 18:335-341, 2006.    -   Navarro C L, Cadinanos J, De Sandre-Giovannoli A, Bernard R,        Courrier S, Boccaccio I, Boyer A, Kleijer W J, Wagner A,        Giuliano F, Deemer F A, Freije J M, Cau P, Hennekam R C,        Lopez-Otin C, Badens C, Levy N. Loss of ZMPSTE24 (FACE-1) causes        autosomal recessive restrictive dermopathy and accumulation of        Lamin A precursors. Hum Mol Genet 14:1503-1513, 2005.    -   Navarro C L, De Sandre-Giovannoli A, Bernard R, Boccaccio I,        Boyer A, Genevieve D, Hadj-Rabia S, Gaudy-Marqueste C, Smitt H        S, Vabres P, Faivre L, Verbes A, Van Essen T, Flori E, Hennekam        R, Beemer F A, Laurent N, Le Merrer M, Cau P, Levy N. Lamin A        and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and        identify restrictive dermopathy as a lethal neonatal        laminopathy. Hum Mol Genet 13:2493-2503, 2004.    -   Padiath Q S, Saigoh K, Schiffmann R, Asahara H, Yamada T,        Koeppen A, Hogan K, Ptacek L J, Fu Y H. Lamin B1 duplications        cause autosomal dominant leukodystrophy. Nature Genet        38:1114-1123, 2006.    -   Pendas A M, Zhou Z, Cadinanos J, Freije J M, Wang J, Hultenby K,        Astudillo A, Wernerson A, Rodriguez F, Tryggvason K,        Lopez-Otin C. Defective prolamin A processing and muscular and        adipocyte alterations in Zmpste24 metalloproteinase-deficient        mice. Nat Genet 31:94-99, 2002.    -   Reid T S, Terry K L, Casey P J, Beese L S. Crystallographic        analysis of CaaX prenyltransferases complexed with substrates        defines rules of protein substrate selectivity. J Mol Biol        343:417-433, 2004.    -   Robber R A, Weber K, Osborn M. Differential timing of nuclear        lamin A/C expression in the various organs of the mouse embryo        and the young animal: a developmental study. Development        105:365-378, 1989.    -   Scaffidi P, Misteli T. Lamin A-dependent nuclear defects in        human aging. Sciencexpress, 27 Apr. 2006.    -   Scaffidi P, Misteli T. Reversal of the cellular phenotype in the        premature aging disease Hutchinson-Gilford progeria syndrome.        Nat Med 11:440-445, 2005.    -   Scaffidi P, Misteli T. Reversal of the cellular phenotype in the        premature aging disease Hutchinson-Gilford progeria syndrome.        Nature Med 11:440-445, 2005.    -   Shelton K R, Egle P M, Cochran D L. Nuclear envelope proteins:        identification of lamin B subtypes. Biochem Biophys Res Comm        103:975-981, 1981.    -   Shumaker D K, Kuczmarski E R, Goldman R D. The nucleoskeleton:        lamins an actin are major players in essential nuclear        functions. Curr Op Cell Biol 15:358-366, 2003.    -   Stewart C, Burke B. Teratocarcinoma stem cells and early mouse        embryos contain only a single major lamin polypeptide closely        resembling lamin B. Cell 51:383-392, 1987.    -   Takedaa M, Noshiroa R, Onozatob M L, Tojob A, Hasannejada H,        Huangc X L, Narikawac S, Endoua H. Evidence for a role of human        organic anion transporters in the muscular side effects of        HMG-CoA reductase inhibitors. Eur J Pharm 483:133-138, 2004.    -   Toth J I, Yang S H, Qiao X, Beigneux A P, Gelb M H, Moulson C L,        Miner J H, Young S G, Fong L G. Blocking protein        farnesyltransferase improves nuclear shape in fibroblasts from        humans with progeroid syndromes. Proc Natl Acad Sci USA        102:12873-12878, 2005.    -   Tsai M Y, Wang S, Heidinger J M, Shumaker D K, Adam S A, Goldman        R D, Zheng Y. A mitotic lamin B matrix induced by RanGTP        required for spindle assembly. Science 311:1887-1893, 2006.    -   Varela I, Cadinanos J, Pendas A M, Gutierrez-Fernandez A,        Folgueras A R, Sanchez L M, Zhou Z, Rodriguez F J, Stewart C L,        Vega J A, Tryggvason K, Freije J M, Lopez-Otin C. Accelerated        ageing in mice deficient in Zmpste24 protease is linked to p53        signalling activation. Nature 437:564-568, 2005.    -   Vergnes L, Peterfy M, Bergo M O, Young S G, Reue K. Lamin B1 is        required for mouse development and nuclear integrity. Proc Natl        Acad Sci USA 101:10428-10433, 2004.    -   Winter-Vann A M, Casey P J. Post-prenylation-processing enzymes        as new targets in oncogenesis. Nat Rev Cancer 5:405-412, 2005.    -   Wydner K L, McNeil J A, Lin F, Worman H J, Lawrence J B.        Chromosomal assignment of human nuclear envelope protein genes        LMNA, LMNB1 and LBR by fluorescence in situ hybridization.        Genomics 32:474-478, 1996.    -   Young S G, Meta M, Yang S H, Fong L G. Prelamin A farnesylation        and progeroid syndromes. J Biol Chem 281:39741-39745, 2006.    -   Zastrow M S, Vlcek S, Wilson K L. Proteins that bind A-type        lamins: integrating isolated clues. J Cell Sci 117:979-987,        2004.    -   Zhang F L, Casey P J. Protein prenylation: molecular mechanisms        and functional consequences. Annu Rev Biochem 65:241-269, 1996.

The invention claimed is:
 1. A method of treating Progeria orrestrictive dermopathy, the method comprising administering to a subjectin need thereof, a pharmaceutical composition comprising asynergistically effective amount of a combination of an inhibitor ofhydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and an inhibitor offarnesylpyrophosphate synthase, wherein the inhibitor of HMG-CoA isselected from the group consisting of pravastatin, atorvastatin,simvastatin, rivastatin, mevastatin, fluindostatin, velostatin,fluvastatin, dalvastatin, cerivastatin, pentostatin, rosuvastatin,pitavastatin, lovastatin, and a pharmaceutically acceptable saltthereof, and the inhibitor of farnesylpyrophosphate synthase is selectedfrom the group consisting of: aledronic acid or its ionic form,alendronate; clodronic acid or its ionic form, clodronate; etidronicacid or its ionic form, etidronate; ibandronic acid or its ionic form,ibandronate; medronic acid or its ionic form, medronate; neridronic acidor its ionic form, neridronate; olpadronic acid or its ionic form,olpadronate; pamidronic acid or its ionic form, pamidronate; risedronicacid or its ionic form, risedronate; tiludronic acid or its ionic form,tiludronate; 4-N,N-dimethylaminomethane diphosphonic acid or its ionicform; dimethylaminomethanediphosphonate; and α-amino-(4hydroxybenzylidene) diphosphonate.
 2. A method of inhibiting theabnormal accumulation of farnesylated and/ geranylgeranylated protein incells of a subject, the method comprising administering to said subjecta pharmaceutical composition comprising a synergistically effectiveamount of a combination of an inhibitor ofhydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and an inhibitor offarnesylpyrophosphate synthase, wherein the inhibitor of HMG-CoA isselected from the group consisting of pravastatin, atorvastatin,simvastatin, rivastatin, mevastatin, fluindostatin, velostatin,fluvastatin, dalvastatin, cerivastatin, pentostatin, rosuvastatin,pitavastatin, lovastatin, and a pharmaceutically acceptable saltthereof, and the inhibitor of farnesylpyrophosphate synthase is selectedfrom the group consisting of: aledronic acid or its ionic form,alendronate; clodronic acid or its ionic form, clodronate; etidronicacid or its ionic form, etidronate; ibandronic acid or its ionic form,ibandronate; medronic acid or its ionic form, medronate; neridronic acidor its ionic form, neridronate; olpadronic acid or its ionic form,olpadronate; pamidronic acid or its ionic form, pamidronate; risedronicacid or its ionic form, risedronate; tiludronic acid or its ionic form,tiludronate; 4-N,N-dimethylaminomethane diphosphonic acid or its ionicform; dimethylaminomethanediphosphonate; and α-amino-(4hydroxybenzylidene) diphosphonate.
 3. The method of claim 1, whereinthe inhibitor of HMG-CoA is pravastatin or a pharmaceutically acceptablesalt thereof, and the inhibitor of farnesylpyrophosphate synthase isaledronic acid or its ionic form, alendronate, or a physiologicallyacceptable salt thereof.
 4. The method of claim 2, wherein the inhibitorof HMG-CoA is pravastatin or a pharmaceutically acceptable salt thereof,and the inhibitor of farnesylpyrophosphate synthase is aledronic acid orits ionic form, alendronate, or a physiologically acceptable saltthereof.
 5. A method of inhibiting the abnormal accumulation offarnesylated and/or geranylgeranylated protein in cells of a subjecthaving progeria or restrictive dermopathy and presenting with signs ofpremature aging but not presenting with high LDL cholesterol levels, themethod comprising administering to said subject a pharmaceuticalcomposition comprising an effective amount of a combination of aninhibitor of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and aninhibitor of farnesylpyrophosphate synthase, wherein the inhibitor ofHMG-CoA is selected from the group consisting of pravastatin,atorvastatin, simvastatin, rivastatin, mevastatin, fluindostatin,velostatin, fluvastatin, dalvastatin, cerivastatin, pentostatin,rosuvastatin, pitavastatin, lovastatin, and a pharmaceuticallyacceptable salt there of and the inhibitor of farnesylpyrophosphatesynthase is selected from the group consisting of: aledronic acid or itsionic form, alendronate; clodronic acid or its ionic form, clodronate:etidronic acid or its ionic form, etidronate; ibandronic acid or itsionic form ibandronate; medronic acid or its ionic form, medronate;neridronic acid or its ionic form, neridronate; olpadronic acid or itsionic form, olpadronate; pamidronic acid or its ionic form, pamidronate;risedronic acid or its ionic form, risedronate; tiludronic acid or itsionic form, tiludronate; 4-N,N-dimethylaminomethane diphosphonic acid orits ionic form; dimethylaminomethanediphosphonate; and α-amino-(4hydroxybenzylidene) diphosphonate.